CONSTRUCTION OF A PHYSICAL MAP OF HUMAN CHROMOSOME 21

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The overall goal of this project is to generate a complete physical map of at least the long arm of human chromosome 21. This chromosome is important for several human genetic diseases, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, progressive myoclonus epilepsy, and probably leukemia. A set of yeast artificial chromosomes (YACs) covering about 20% of the long arm of chromosome 21 (21q) already has been generated in this laboratory on behalf of the chromosome 21 Joint YAC Screening Effort. Moreover, this laboratory has constructed a pulsed field gel map of 2lq spanning over 44 million base pairs of DNA, or over 90% of the estimated size of this chromosome arm. Reagents available to construct the map, in the form of a set of ordered, overlapping YACs spanning the chromosome include 3 different YAC libraries representing roughly 13-fold coverage of the human genome, 107 additional regionally mapped chromosome 21-specific DNA probes not yet used to screen YAC libraries, a very detailed Chinese hamster/human cell hybrid regional mapping panel for chromosome 21 generated and characterized in this laboratory and 2 microclone libraries from microdissected chromosome 21s comprising at least 740,000 chromosome 21 clones. Methods in place to be used include both PCR and hybridization based YAC library screening protocols, YAC end cloning by Vectorette PCR and alu-vector PCR, long-range restriction mapping of YACs, and fluorescence in situ hybridization (FISH) to metaphase chromosomes using YACs as probes to assess YAC chimerism. A new technique, RecA Assisted Restriction Endonuclease (RARE) Cleavage, will be assessed to aid in validating the fidelity of the YAC contig, filling in gaps (closure), and for other mapping purposes.